Use of hydrogels for biosensors having elevated sensitivity

ABSTRACT

The present invention relates to measures for determining glucose and for diagnosing diseases based on impaired glucose metabolism. In particular the present invention relates to the use of a polymer-based hydrogel consisting of at least one water-soluble polymer in a sensor for enriching a dissolved glucose-binding protein, wherein the at least one water-soluble polymer is capable of interacting with the glucose-binding protein and wherein the glucose-binding protein is present in a complex with a ligand.

The present invention relates to the use of a polymer-based hydrogelcomprising at least one water-soluble polymer in a sensor for enrichinga dissolved glucose-binding protein, wherein the at least onewater-soluble polymer is capable of interacting with the glucose-bindingprotein and wherein the glucose-binding protein is present in a complexwith a ligand.

The determination of the concentration of glucose by efficient andreliable measurement technology is of great importance in many fields oftechnology. Not only in pure laboratory analysis, but also in thefoodstuffs industry, e.g. in the field of oenology and in the medicalfield, for example in the diagnosis of diseases which are caused byimpaired glucose metabolism, e.g. diabetes mellitus or metabolicsyndrome, the rapid and reliable determination of the glucoseconcentration in given solutions is of central importance. In the fieldof the diagnosis of diseases that are based on impaired glucosemetabolism, glucose sensors are used both in devices which areimplantable into the body and can measure the glucose content in asample taken within the body, and also in sensors that measure theglucose content ex vivo from a test subject's sample.

For determining the glucose content in a solution, systems made ofsensor molecule and ligand have been described, wherein the sensor is aglucose-binding protein and the ligand a competitor for glucose, whichis initially present in the sensor bound to the glucose sensor molecule.By competition with glucose during the measurement procedure, thecompetitor is displaced from the glucose-binding protein. Thedisplacement of the competitor from the glucose-binding protein by theglucose during this can be detected by means of a change in a physicalor chemical property of the molecules, e.g. by means of fluorescenceresonance energy transfer (FRET). The aforesaid systems must of coursebe present in a spatially demarcated region of the sensor.

For enclosure of the system components, i.e. of the glucose-bindingmolecule and the ligand, in an aqueous medium, semi-permeable membranesare used. Such membranes can for example consist of regeneratedcellulose, polyethylene glycol, polyurethane, layer-by-layer (LBL)layers, polyether sulfones, parylene layers or perforated silica (e.g.US2007/0122829). Alternatively, hydrogels, which are however usuallyalso enclosed by an additional membrane, can also be used (see e.g. U.S.Pat. No. 6,485,703; US2007/0105176).

However, the glucose sensors described in the state of the art exhibitrelatively low glucose activity. The glucose activity and hence thesensor performance is predominantly determined by the binding constantsfor the complex of glucose-binding protein and competitor andglucose-binding protein and glucose. Furthermore, the mobility of thesensor components also plays a part. In hydrogel matrices, this israther restricted (Rounds 2007, J. Fluoresc. 17: 57-63).

With in vivo sensors for measuring glucose, the sensor must constantlyreact reversibly to changes in the analyte concentration. Thus thebinding constant must not be too high, since otherwise the sensor wouldalready be saturated at low analyte concentrations, and would no longerbe able to indicate concentration changes. Moreover, a further problemof the in vivo sensors consists in that the analyte concentration rangeis fixed and cannot be optimized by dilution or concentration.Essentially, in the state of the art, systems consisting of ligand andglucose-binding protein are described wherein medium binding constantsare implemented. Adaptation of the measurement sensitivities byalteration of the concentrations of either glucose-binding protein orligand or both (e.g. by enrichment) are as a rule limited by the lowsolubility of the respective molecules. Hence in the sensors describedin the aforesaid state of the art, both the measurement sensitivity andalso the measurement precision of the sensor are limited.

The purpose of the present invention is to provide measures which makeit possible to improve the measurement sensitivity and precision ofbiosensors for measurement of the glucose level. The invention is solvedby the embodiments described by the claims and the embodiments which aredisclosed below.

The present invention thus relates to the use of a polymer-basedhydrogel comprising at least one water-soluble polymer in a sensor forenriching a dissolved glucose-binding protein, wherein the at least onewater-soluble polymer is capable of interacting with the glucose-bindingprotein and wherein the glucose-binding protein is present in a complexwith a ligand.

The term “hydrogel” describes a water-containing polymer whose moleculesare chemically or physically linked into a three-dimensional network.The polymer molecules can be linked together into the three-dimensionalnetwork by covalent or ionic bonds or by entanglement or interweaving.The polymers which form the hydrogel preferably contain hydrophilicpolymer components which enable the uptake of aqueous solutions, andgroups which are capable of interacting with the glucose-bindingprotein. In particular for lectins such as concanavalin A, as well as abinding site for monosaccharides, e.g. glucose, a further binding sitefor terminal sugar molecules has also been described (Moothoo 1999,Glycobiology 9(6): 539-545; Bryce 2001, Biophysical Journal 81:1373-1388; Moothoo 1998, Glycobiology 8(2): 173-181; Delatorre 2007, BMCStructural Biology 7:52). Sugar or structurally similar molecules orresidues in the polymer molecules of the hydrogel are preferably capableof interacting with this further binding site. Depending on the natureof the glucose-binding protein, according to the invention a polymer isselected which has residues which are capable of interacting with theglucose-binding protein, without thereby blocking the binding site forglucose. Suitable residues for this can also be antibodies or antibodyfragments, such as Fab F(ab)₂, scF_(V) or the like, which are coupled tothe polymer, and which specifically recognize the glucose-bindingprotein. Aptamers can also be used instead of antibodies in an analogousmanner, which specifically recognise the glucose-binding protein.

Preferably the at least one polymer is selected from the groupconsisting of: alginates, sepharose, hyaluronic acid, chitosan,caragenans, polyvinyl alcohols (PVAs), polyethylene glycols (PEGs),poly(2-oxazolines), polyacrylamides (e.g. dimethylacrylamide),polyhydroxyacrylates (e.g. polyhydroxymethacrylate,polyhydroxyacrylamides, polyvinylpyrolinones),(2-methyl-3-ethyl[2-hydroxyethyl]) polymers, polyhydroxyalkanoates(PHAs), poly(2-methyl-2 oxazolines), poly(2-ethyl-2 oxazolines),poly(2-hydroxyethyl-2 oxazolines), poly(2-(1-(hydroxymethyl)-ethyl)-2oxazolines), poly-(hydroxyethyl methacrylate) (PHEMA),poly-(hydroxyethyl acrylate) (PHEA), poly-vinylpyrolidones,poly-(dimethyl)acrylamide, poly-(hydroxyethyl)acrylamide, polyvinylalcohols (including copolymers with vinyl acetates and/or ethylene),poly(ethylene-co-vinyl alcohol), poly(vinyl acetate-co-vinyl alcohol),poly(ethylene-co-vinyl acetate-co-vinyl alcohol), polyethylene glycolsand poly(ethylene glycol-co-propylene glycol).

Particularly preferably, the at least one polymer is selected from thegroup consisting of: alginates, sepharose, hyaluronic acid, chitosan,and caragenans. Quite particularly preferably, the at least one polymeris an alginate.

It goes without saying that the hydrogel in the sensor can contain yetfurther polymers. In particular, the polymer-based hydrogel can comprisea first hydrogel matrix made of alginate and a second hydrogel matrixwhich is capable of forming an interpenetrating network within the firsthydrogel matrix. This second hydrogel matrix preferably consists of awater-soluble polymer having at least one crosslinkable group permolecule and a molecular weight of at most 500,000. Particularlypreferable is a molecular weight of at most 250,000, 200,000, 150,000,100,000 or 50,000. In this, the polymer of the second hydrogel matrix ispreferably selected from the group consisting of: polyvinyl alcohols(PVAs), polyethylene glycols (PEGs), poly(2-oxazolines), polyacrylamides(e.g. dimethylacrylamide), polyhydroxyacrylates (e.g.polyhydroxymethacrylate, polyhydroxyacrylamide, polyvinylpyrolinones),(2-methyl-3-ethyl[2-hydroxyethyl]) polymers, polyhydroxyalkanoates(PHAs), poly(2-methyl-2 oxazolines), poly(2-ethyl-2 oxazolines),poly(2-hydroxyethyl-2 oxazolines), poly(2-(1-(hydroxymethyl)-ethyl)-2oxazolines), poly-(hydroxyethyl methacrylate) (PHEMA),poly-(hydroxyethyl acrylate) (PHEA), polyvinylpyrolidones,poly-(dimethyl)acrylamide, poly-(hydroxyethyl)acrylamide, polyvinylalcohols (including copolymers with vinyl acetates and/or ethylene),poly(ethylene-co-vinyl alcohol), poly(vinyl acetate-co-vinyl alcohol),poly(ethylene-co-vinyl acetate-co-vinyl alcohol), polyethylene glycolsand poly(ethylene glycol-co-propylene glycol).

Particularly preferably, the second hydrogel matrix is formed ofpolyvinyl alcohol. Particularly preferred is polyvinyl alcohol having amolecular weight of 10,000 to 100,000, more preferably 10,000 to 50,000,more preferably 10,000 to 20,000, and quite particularly preferably15,000. Particularly preferably, the polyvinyl alcohol has a crosslinkercontent of at most 0.5 mmol/g, 0.4 mmol/g, 0.35 mmol/g, or 0.3 mmol/gand quite particularly preferably 0.35 mmol/g. Furthermore, thepolyvinyl alcohol particularly preferably has a prepolymer solidscontent of less than 40 weight percent.

As well as the first hydrogel matrix and the second hydrogel matrix, thehydrogel according to the invention can contain additives, e.g.stabilizers, emulsifiers, antioxidants, UV stabilizers, detergentsand/or UV initiators.

The hydrogel used according to the invention can moreover be enclosed bya further, preferably semipermeable, covering material. By means of thisenclosure, “leaching” of the sensor components from the hydrogel isprevented. Possible covering materials are semipermeable membranes orother hydrogel matrices. Semipermeable membranes can preferably consistof regenerated cellulose, polyethylene glycol, polyurethane,layer-by-layer (LBL) layers, polyether sulfones, parylene layers orperforated silica. However, a further hydrogel matrix is also possibleas covering material. Preferably, this can be formed from a polymerselected from the group consisting of alginates, sepharoses, hyaluronicacid, chitosan, caragenans, polyvinyl alcohols (PVAs), polyethyleneglycols (PEGs), poly(2-oxazolines), polyacrylamides (e.g.dimethylacrylamide), polyhydroxyacrylates (e.g. polyhydroxymethacrylate,polyhydroxyacrylamides, polyvinylpyrolinones),(2-methyl-3-ethyl[2-hydroxyethyl]) polymers, polyhydroxyalkanoates(PHAs), poly(2-methyl-2 oxazolines), poly(2-ethyl-2 oxazolines),poly(2-hydroxyethyl-2 oxazolines), poly(2-(1-(hydroxymethyl)-ethyl)-2oxazolines), poly-(hydroxyethyl methacrylate) (PHEMA),poly-(hydroxyethyl acrylate) (PHEA), poly-vinylpyrolidones,poly-(dimethyl)acrylamide, poly-(hydroxyethyl)acrylamide, polyvinylalcohols (including copolymers with vinylacetates and/or ethylene),poly(ethylene-co-vinyl alcohol), poly(vinyl acetate-co-vinyl alcohol),poly(ethylene-co-vinyl acetate-co-vinyl alcohol), polyethylene glycolsand poly(ethylene glycol-co-propylene glycol).

The term “glucose-binding protein” in the context of the inventionrelates to proteins which are capable of interacting specifically withglucose. Whether a protein is capable of interacting specifically withglucose can readily be determined by those skilled in the art by bindingtests known in the state of the art. Preferably, the glucose-bindingprotein is also capable of interacting with the polymer of the hydrogel.Preferably, this interaction is not mediated by the glucose-binding siteof the glucose-binding protein. Particularly preferably, theglucose-binding protein is selected from the group consisting oflectins, enzymes which bind glucose as substrate, and antibodies whichspecifically recognize glucose. The term also includes surrogatemolecules which can specifically recognize glucose, preferably aptamerswhich specifically recognize glucose. Quite particularly preferably, theglucose-binding protein is concanavalin A.

Nucleic acid sequences and amino acid sequences which encode theaforesaid glucose-binding proteins are known in the state of the art(Yamauchi 1990, FEBS Letters 260(1): 127-130). Accordingly, theaforesaid proteins can readily be prepared by those skilled in the art.Said proteins can for example be prepared recombinantly or be purifiedfrom a biological source. Furthermore, the proteins can also bechemically synthesized. Moreover, most of the aforesaid proteins arecommercially available. Antibodies or aptamers which specificallyrecognize glucose can readily be prepared by those skilled in the art bymethods for antibody or aptamer obtention known in the state of the art.

The aforesaid glucose-binding proteins and in particular concanavalin Acan preferably also have chemical modifications which mediate increasedwater solubility compared to unmodified versions of the glucose-bindingproteins. Such modifications preferably comprise functionalization witha water-soluble polymer and in a particularly preferable embodiment canbe selected from the group consisting of: pegylation, acetylation,polyoxazolinylation and succinylation.

The detection of glucose in a sample for analysis is effected in thedevice according to the invention by displacement of the ligand bound tothe glucose-binding protein by the glucose contained in the sample(competition between ligand and glucose). Hence the ligand preferablyhas a lower affinity to the glucose-binding protein than glucose. Thedisplacement can preferably be detected by labeling of the ligand with adye or another detectable marker molecule. Dyes or other markermolecules which on approach of the molecules bound to them cause achange in at least one measurable physical or chemical property haveproved particularly suitable for the detection of a displacement.Suitable systems include those in which a measurable signal is eithersuppressed or generated by means of energy transfer between the dyemolecules. Such energy transfer-based systems are for example describedin more detail in WO2001/13783. These can preferably be systems in whicha fluorescence signal is suppressed by quenching effects when the dye ormarker molecules—and hence the glucose binding protein and itsligand—are in spatial proximity. After the displacement of the ligand bythe analyte, the quenching effect is then canceled. This effect isdetectable by a change in the fluorescence. For the detection, forexample fluorescence photometers as described in WO2002/087429 can beused. Other suitable systems are so-called fluorescence resonance energytransfer (FRET)-based detection systems. In these, two interactingcomponents, such as the glucose-binding protein and its ligand, arelabeled with fluorescent dyes. One component is coupled with an acceptordye, the other with a donor dye. Through the interaction of thecomponents, the dyes come into spatial proximity, whereby the FRETeffect is produced. In this, excitation energy is transferred from thedonor to the acceptor dye and thus the intensity of the donor dye ismeasurably decreased. As soon as the interaction of the components isinterrupted, the fluorescent intensity of the donor again increases. Inthe case of the device according to the invention, the glucose can thusbe detected via the increase in the intensity of a signal which isgenerated by a dye or marker molecule after separation of the complex ofglucose-binding protein and ligand by the analyte. The dye or the markermolecule is coupled either to the glucose-binding protein or the ligand.For example, a donor dye can be coupled to the glucose-binding proteinor the ligand, while the component not coupled to the donor dye iscoupled to a suitable acceptor dye. In a thus configured deviceaccording to the invention, the FRET effect as a result of the bindingof the ligand to the glucose-binding protein can be observed beforeexposure to a glucose-containing sample. After exposure, the glucosedisplaces the ligand, so that the measurable intensity of thefluorescence of the donor dye increases, in fact proportionately to thequantity of glucose.

Birch et al. (Birch 2001, Spectrochimica Acta Part A 57: 2245-2254) havecalculated the mathematical solution of the chemical equilibrium for thecase of concanavalin A as glucose-binding protein and dextran as ligand.Simulations with varying concanavalin A and dextran concentrations haveshown that the ratio (dextran)/(Con A-dextran complex) depends onlyslightly on the starting concentrations and that the binding constantsK_(Dex) and K_(Gluc) are the main factors for sensor performance.

Surprisingly, however, the structure of the dyes also affects theglucose activity. Thus according to the invention a combination of arhodamine and an oxazine dye is markedly superior to the conventionallyused combination of a xanthene and a rhodamine dye (FITC-TMR).

Preferably, the glucose-binding protein which is used in the context ofthe use according to the invention is linked to an oxazine dye. How sucha linkage can be effected is well known to those skilled in the art andadequately described in the state of the art. Particularly preferably,the oxazine dye is an oxazine acceptor selected from the groupconsisting of: ATTO655, ATTO680, EVOblue10, EVOblue30, EVOblue90 andEVOblue100. Quite particularly preferably, ATTO680 is used. Said oxazinedyes are commercially available.

The preferred degree of labeling (DOL) for the glucose-binding protein,e.g. concanavalin A, is 0.1 to 4, more preferably 1 to 4 andparticularly preferably 1 to 3. With concanavalin A, a DOL of 1 herecorresponds to one mol of dye per mole of concanavalin A tetramer(MW=104,000). With a high DOL and use of relatively nonpolar dyes(typically long wavelength fluorescent dyes) the glucose-binding proteinis preferably functionalized with PEG. The preferred degree ofpegylation is 0.1 to 5, preferred molecular weight 200 to 10,000,particularly preferably 800 to 8000, still more preferably 800 to 5000.

In the context of the experiments on which the present invention isbased, it was established that chemical modifications of theglucose-binding proteins which mediate increased water-solubility incomparison to unmodified forms can advantageously be used in order toachieve a better degree of labeling on the glucose-binding proteins. Forthe modified glucose-binding proteins according to the presentinvention, higher degrees of labeling with a dye can also be achievedthan for unmodified forms thereof. For such modified concanavalin Aproteins, concentrations preferably greater than 0.5 mg/(g matrix) andquite particularly preferably between 2 and 60 mg/(g matrix) can beachieved. Surprisingly, the measured glucose activity of modifiedconcanavalin A proteins here was also comparable with that of nativeconcanavalin A in hydrogel.

In this, the dissolved glucose-binding protein can be present in thehydrogel at an at least 5-fold, at least 10-fold, at least 15-fold or atleast 20-fold elevated concentration compared with an aqueous solution.

The improved solubility is particularly relevant when theglucose-binding proteins are to be labeled with a dye, sinceglucose-binding proteins labeled with dyes, for example a concanavalin Amodified with one of the aforesaid oxazine dyes, have still furtherreduced solubility in aqueous solution. Here, the higher the degree oflabeling is, the lower is the solubility in aqueous solution. However,it is precisely a higher degree of labeling that is needed forglucose-binding proteins as sensor components in the devices accordingto the invention.

The term “ligand of the glucose-binding protein” means a molecule whichis capable of entering into specific bonding with the glucose-bindingprotein, during which the molecule essentially interacts with the samebinding site as glucose, so that the bound molecule can be displacedfrom the binding site on the glucose-binding protein by glucose.Molecules suitable as the ligand are therefore structurally related toglucose. The ligand of the glucose-binding protein is preferably anoligosaccharide, a glycosylated macromolecule, e.g. a glycosylatedprotein or peptide, or a glycosylated nanoparticle. The aforesaidmolecules which can be used as ligands of the glucose-binding proteinare known in the state of the art and can readily be prepared by thoseskilled in the art. Particularly preferably, a dextran is used as ligandof the glucose-binding protein.

The ligand of the glucose-binding protein in the sensor is preferablycoupled with a rhodamine dye. Particularly preferably, ATTO590, ATTO610,ROX, TMR, rhodamine G6, Alexa Fluor rhodamine dyes or Dy590, and quiteparticularly preferably ATTO590, can be used in this.

The preferred degree of labeling (DOL) for the ligand of theglucose-binding protein, e.g. dextran, is 0.00003 to 0.017 (moldye)/(mol subunit), particularly preferably 0.0005 to 0.007 (moldye)/(mol subunit) and quite particularly preferably 0.001 to 0.0016(mol dye)/(mol subunit). The degree of labeling of the ligand also hasan influence on the glucose activity. Excessively low, just likeexcessively high, degrees of labeling lead to poor glucose activity.

In a preferred embodiment of the use according to the invention, theglucose-binding protein is concanavalin A. Likewise, through chemicalmodifications, preferably pegylation, acetylation, polyoxazolinylationor succinylation, the concanavalin A exhibits increased water-solubilitycompared to the unmodified concanavalin A. In a preferred embodiment,the glucose-binding protein is linked to an oxazine dye and the ligandof the glucose-binding protein to a rhodamine dye. Quite particularlypreferably, a concanavalin A/dextran system is used in the sensordevice, wherein the dextran is linked to a rhodamine donor dye and theconcanavalin A to an oxazine acceptor dye. In the preferred concanavalinA/dextran system, the components are preferably present in a mass ratio(dextran/Con A) from 1:1 to 1:40, with mass ratios close to 1:10 beingparticularly preferable.

In the context of the present invention, it was ascertained that thecombination of oxazine and rhodamine dyes causes a heterodimericinteraction between the dye residues, which intensifies the glucoseactivity. Thus through the use of oxazine acceptor and rhodamine donordyes, a 2.1-fold glucose activity can preferably already be achieved inaqueous solution. In the hydrogel used in the context of the presentinvention, a 2.6-fold increase in the glucose activity could even beproduced.

Through the use of a polymer which is capable of interacting with theglucose-binding protein, a higher concentration of glucose-bindingprotein and ligand in the hydrogel can be achieved. This increases themeasurement sensitivity but also the measurement precision of thesensor, in particular with in vivo applications with given glucoseconcentration.

In the context of the present invention, it was advantageouslyascertained that the use of a hydrogel consisting of a first hydrogelmatrix made of alginate and a second hydrogel matrix which forms aninterpenetrating network within the first is capable of creating anenvironment for the sensor components, namely the glucose-bindingprotein and the competitive ligands of the glucose-binding protein,which allows efficient determination of the glucose activity. Manyapproaches for glucose measurement by means of fluorescence aresuccessful in solution, but lose their activity when the sensorcomponents are embedded in hydrogel matrices, since the mobility of thesensor components is restricted (Rounds 2007, J. Fluoresc. 17: 57-63; US2007/0105176 A1). Surprisingly, however, particularly also in view ofthe calculations of Birch et al. (Birch 2001, loc cit.), activityincreased by up to 2.6-fold compared to aqueous solutions could bedetected in the present case. Through the selection of suitable hydrogelmatrices, an enrichment of the glucose-binding protein far above itssolubility limit in aqueous solution could be achieved. In the case ofconcanavalin A, for example a more than 10-fold increased concentrationcompared to a concentration achievable in free solution could beachieved. Usually, the solubility of the receptor component in freesolution is further adversely influenced by the addition of the ligand,since the receptor/competitor complex exhibits a lower solubility owingto its size and often also owing to multivalences. While for example aconcanavalin A/dextran complex in solution at a mass ratio of 1:10already begins to precipitate beyond a concanavalin A concentration of0.5 mg/(g solution), in a suitable hydrogel with the same mass ratio,concanavalin A concentrations of over 50 mg/(g matrix) can be prepared.Hence the usable concentration range can be extended 100-fold. This isof particular importance for applications wherein the analyteconcentration is fixed and cannot be adjusted by dilution orconcentration. Precisely such difficulties arise with in vivoapplications such as the determination of the glucose level in bodyfluids. In the in vivo situation, the concentration of the analyteglucose cannot be adjusted to the specific assay conditions, but mustrather be taken as given.

A further solubility problem arises in particular with in vivoapplications of biological sensors. Owing to the higher wavelength, theintrinsic fluorescence of the tissue declines, so that with in vivoapplications long wavelength fluorescent dyes are used. However, thesefluorescent dyes are typically apolar owing to their molecular structureand size (conjugated systems). If the glucose-binding protein is onlylabeled with such a dye, the solubility further decreases, so that highdegrees of labeling are also not possible. As already described above,it can therefore be necessary to functionalize the glucose-bindingprotein with e.g. polyethylene glycol, in order to enable increasedsolubility and higher degrees of labeling associated therewith. However,functionalization with polyethylene glycol (pegylation) as a rule leadsto a markedly reduced glucose activity of for example nativeconcanavalin A (relative glucose activity of 0.4 or less). Surprisingly,in the context of the present invention it was established that in thehydrogel of the sensor device this worsening does not occur. Rather,glucose activities for concanavalin A functionalized with polyethyleneglycol can be achieved which are comparable with those of nativeconcanavalin A (relative glucose activity=2.2 or 2.6). Furthermore, itwas found that the glucose activity can be still further raised byincreasing the degree of labeling on the concanavalin A in a hydrogelsuch as is used in the device of the present invention. Thus,surprisingly, in the enriching hydrogel, by increasing the degree oflabeling on the concanavalin A functionalized with polyethylene glycol,a doubling of the glucose activity could even be achieved. Compared tothe measurement in solution, the glucose activity in the hydrogel usedaccording to the invention in the sensor device is even increased4.3-fold. The use of the hydrogel in the device according to theinvention thus makes it possible to prepare concentration ratios for theglucose-binding protein and its ligand which with equivalent degrees oflabeling allow glucose activities which are increased 4-fold compared toconcentrations which are preparable in aqueous solutions. Thisadvantageously also enables the use of the device according to theinvention under conditions wherein the analyte concentration cannot beadapted to the assay conditions, such as with in vivo applications.

In the context of the use according to the present invention, the sensorcan preferably be used for determining glucose content.

Thereby, a sample can be tested. In the context of the presentinvention, the term “sample” should be understood to mean a composition,preferably an aqueous composition, which presumably or actually containsglucose. The sample is preferably a biological sample. Quiteparticularly preferably, the sample is a body fluid, in particulartissue fluid (interstitial fluid), blood, plasma, serum, lymph, saliva,tear fluid, sweat or urine. Particularly preferably, the sample istissue fluid, blood, serum or plasma.

Provided that the sample is a biological material, e.g. a body fluid, itcan preferably be obtained from a test subject who either actually orpresumably has impaired glucose metabolism. Preferably here, theimpaired glucose metabolism is caused by diabetes mellitus or metabolicsyndrome.

The sensor can thus also be used in the context of the use according tothe invention for ex vivo diagnosis of diseases or impairments of theglucose metabolism. In particular, the sensor can be used for thediagnosis of impaired glucose metabolism and connected therewith ofdiseases such as diabetes mellitus or metabolic syndrome, or fordetermining the need for a therapeutic measure in a patient withimpaired glucose metabolism.

The aforesaid therapeutic measures comprise those which are used for thetreatment of diabetes mellitus or metabolic syndrome. As well as theadministration of drugs, e.g. insulin, this also includes theimplementation of other therapeutic measures concerning which a decisioncan be taken on the basis of the impaired glucose metabolism determined.These include therapeutic interventions, e.g. gastric bypass operations,or changes in lifestyle, e.g. the implementation of special diets. Thesensor can be coupled with a further device, e.g. a device whichcontrols the delivery of a drug. In this, a device which controls thedelivery of insulin is preferable. The delivery of insulin can then becontrolled on the basis of the need determined by the sensor.

For ex vivo use, the sensor from the use according to the invention canfor example be introduced into microtiter plates and anchored there.Samples for assay are then applied into the wells of the microtiterplates and can then be assayed with a reader device. Such an approachenables the simultaneous assay of a large number of samples and is thusalso economical, in particular in clinical diagnostic practice.

Alternatively, the sensor for the in vivo use can be introduced into thebody. Here it should be noted that the measurement of the glucose level,which is of course also the basis for the diagnosis, requires that thedevice come into contact with a body fluid which contains glucose,wherein the concentration of glucose in the fluid is representative ofthe glucose level to be determined. Suitable body fluids are enumeratedat another place in the description. Particularly preferably, the bodyfluid is tissue fluid.

The sensor is preferably introduced at places in the body which allowoptical measurement of the signal generated by the device. Places witheither a small tissue thickness between device and body surface or withtransparent tissues which can be effectively penetrated by the generatedsignal are suitable. Particularly preferably, the device is positionedunder the skin (subcutaneously) or in the eye, e.g. subconjunctivally.Appropriate methods for the implantation of the device are known in thestate of the art.

Alternatively, the signal created by the device according to theinvention can also be transferred outside the body by means of asuitable transfer medium. For this, a signal-conducting material canpreferably be used as flexible cable, e.g. a glass fiber cable. However,the transfer of the signal can also be effected wirelessly, e.g. as aninfrared, radio or wireless signal. It goes without saying that in thiscase the signal created by the device according to the invention mustfirstly be read by a detector which must likewise be installed in thedevice or at least in spatial proximity and be converted into anelectromagnetic signal, for example a wireless signal. Thiselectromagnetic signal can then be received by a receiver lying outsidethe body and evaluated.

Through the use according to the invention of the hydrogel in thesensor, efficient ex vivo and in vivo diagnosis of the blood sugar levelis enabled, and hence the early recognition of diseases which areassociated with impaired glucose metabolism, and also the managementthereof, and the selection of therapeutic measures.

The invention is illustrated by the following practical examples.However, the examples do not limit the protection range.

EXAMPLES Example 1 Preparation of Sensors for Determining GlucosePreparation of Hydrogel Particles (Enriching Matrix)

1 g of sodium alginate is dissolved in 100 g water with stirring. 66.2 gof CaCl2×2H2O are dissolved in 4931.3 g water in a 5 L beaker.

The alginate solution is passed into a dual nozzle via a pump. At thesame time, compressed air is connected to the second inlet of thenozzle, so that the alginate solution is atomized into fine droplets.The droplets are carried by the air flow into a bath containing thecalcium chloride solution, where they gel and sink to the bottom. Thegelled beads are then collected.

Preparation of Sensors in Enriching Matrix:

For loading, alginate beads are successively incubated in a dye-labeledconcanavalin A solution and a dye-labeled dextran solution. The loadedbeads are then centrifuged down and the supernatant solution decantedoff. The loaded beads are optionally then incubated overnight in asolution of a second polymer (e.g. PVA or PEG-based) and optionallyisolated by centrifugation. The beads are then mixed into an aqueoussolution of a photochemically crosslinkable polymer. This mixture isthen crosslinked with UV light in order to prevent the sensor componentsleaching out of the alginate beads.

The quantities for this depend on the concentration of the analyte to bemeasured and the degree of labeling is selected depending on the desiredintensity of the fluorescence signal. As the photochemicallycrosslinkable polymer, for example Nelfilcon polymer, a polyvinylalcohol modified with acrylamide groups, can be used. For thephotochemical crosslinking, 0.1% Irgacure 2959 is also added. Thefinished solution is dispensed into suitable molds and cured with UVlight.

Preparation of Sensors in Non-Enriching Matrix:

Dye-labeled concanavalin A solution and dye-labeled dextran solution aresuccessively fed into a water-based prepolymer mixture and stirred for 3hours. The quantities for this depend on the concentration of theanalyte to be measured and the degree of labeling is selected dependingon the desired intensity of the fluorescence signal. As thephotochemically crosslinkable polymer, for example Nelfilcon polymer, apolyvinyl alcohol modified with acrylamide groups, can be used. For thephotochemical crosslinking, 0.1% Irgacure 2959 is also added. Thefinished solution is dispensed into suitable molds and cured with UVlight.

Example 2 Determination of the Glucose Activity for the SensorsDetermination of the Glucose Activity in Sensors

The fluorescence spectrum of the sensors is determined at variousglucose concentrations. The change in the fluorescence intensities ofthe donor with increasing glucose content serves as a measure of thequality of the glucose sensor. Since with an in vivo glucose sensorglucose concentrations between 50 and 500 mg/dL have to be measured, theglucose activity is calculated as follows:GA=(intensity_(500 mg/dL)−intensity_(50 mg/dL))/intensity_(50 mg/dL)

For better comparison, all response values are normalized to theresponse of the same system in solution. For the determination of therelative glucose activity, the glucose activity of the sensor (inmatrix) is divided by the glucose activity in solution.Rel GA=GA(matrix)/GA(solution)Determination of the Glucose Activity in Solution:

Con A solution and dextran solution are diluted in buffer solution andstirred for several hours. The fluorescence spectrum of the solution isdetermined at various glucose concentrations. The change in thefluorescence intensities of the donor with increasing glucose contentserves as a measure of the quality of the system. Since with an in vivoglucose sensor glucose concentrations between 50 and 500 mg/dL have tobe measured, the glucose activity is calculated as follows:GA=(intensity_(500 mg/dL)−intensity_(50 mg/dL))/intensity_(50 mg/dL)

Example 3 Determination of the Influence of the Matrix

According to the invention, the glucose-binding protein and the ligandare incorporated into a hydrogel matrix which exhibits a certaininteraction with the glucose-binding protein. For this, the hydrogelmatrix is selected such that firstly the interaction of glucose-bindingprotein and hydrogel matrix is greater than that between glucose-bindingprotein and aqueous solution (enrichment of the sensor components). Onthe other hand, however, the interaction between glucose-binding proteinand the analyte (glucose) must be unaffected or not significantlyaffected by the interaction of glucose-binding protein and hydrogelmatrix.

With suitable selection of the hydrogel matrix, through the interactionbetween glucose-binding protein and hydrogel matrix an enrichment of theglucose-binding proteins in the matrix far above the solubility limit ofthe glucose-binding protein in aqueous solution is achieved. In the caseof Con A, for example up to 10 times higher concentrations can beachieved than in free solution. The solubility problems become stillmore extreme after addition of the ligand (e.g. dextran), since theglucose-binding protein-ligand complex exhibits lower solubility owingto its size and often also owing to multivalences. While the ConA-dextran complex in solution at a mass ratio of 1:10 already begins toprecipitate beyond a Con A concentration of 0.5 mg/g, Con Aconcentrations of over 50 mg/g can be prepared in a suitable hydrogelmatrix at the same mass ratio. Through the hydrogel matrix, the usableconcentration range of the glucose-binding protein, e.g. Con A, can beincreased 100-fold.

Since with an in vivo application the range of the analyte concentrationis fixed and cannot be adjusted e.g. by dilution or concentration, theconcentrations of glucose-binding protein and ligand must be adapted tothe in vivo concentration range of the analyte. However, there is oftenthe difficulty that glucose-binding protein concentrations and/or ligandconcentrations which exceed the solubility limit would therefore benecessary.

For example, with the Con A-dextran system, in solution a Con Aconcentration of only 0.5 mg/g can be established at a mass ratio(dex:Con A) of 1:10, since otherwise the Con A-dextran complex begins toprecipitate. Here, for the determination of the relative glucoseactivity (rel GA), the glucose activity (GA) achieved at thisconcentration is set equal to 1. As expected, in a non-enriching matrixthe glucose activity is decreased to half (rel GA=0.52) owing to thelower mobility of the sensor components. In enriching matrix, with thesame concentration almost the same response as in solution is obtained(rel GA=0.83). However, significantly higher Con A concentrations canalso be prepared in the enriching matrix. At a Con A concentration of 10mg/g, a 2.6-fold increased glucose activity is obtained in enrichinghydrogel matrix (see table 1).

TABLE 1 Influence of the matrix in non-enriching in enriching inenriching in solution matrix matrix matrix Con A 0.5 mg/g 0.5 mg/g 0.5mg/g 10 mg/g concentration Rel GA 1 0.52 0.83  2.55 Native Con A withDOL = 1, dextran with 0.001 mol dye (D) per mol dex subunit (SU), massratio dex:Con A = 1:10

Example 4 Determination of the Influence of the Concentration of theReceptor

In enriching matrix, a dependence of the glucose activity on thestarting concentration of the glucose-binding protein and of the ligandis clearly seen. With a rise in the concentration, a rise in the glucoseactivity is also obtained. At very high receptor concentrations, theglucose activity again declines. The optimal Con A concentration for anin vivo assay lies between 8 and 20 mg Con A/g matrix (see table 2).

TABLE 2 Influence of the receptor concentration Con A conc. [mg/g] 0.510.0 13.3 30 52.2 Relative Glucose 0.83 2.55 2.76 2.14 1.76 activity(rel GA) Native Con A with DOL = 1, dextran with 0.001 mol dye (D) permol dex subunit (SU), mass ratio dex:Con A = 1:10

Example 5 Determination of the Influence of the degree of LabelingReceptor

Owing to the decreasing intrinsic fluorescence of the tissue with higherwavelengths, long wavelength fluorescent dyes must be used with in vivoapplications. These fluorescent dyes are typically quite nonpolar owingto the larger conjugated system. If the glucose-binding protein islabeled with such dyes, then its solubility decreases, so that highdegrees of labeling are often not possible owing to precipitation. Inorder to increase the solubility of the glucose-binding protein, it isadvantageous to functionalize this e.g. with polyethylene glycol.Thereby, the solubility of the glucose-binding protein increases, as aresult of which higher degrees of labeling are possible in thesynthesis.

However, pegylated Con As in solution lead to only less than half of theglucose activity of native Con A (rel GA=0.4). Surprisingly, in thehydrogel matrix this worsening does not arise. With PEG-Con A,comparable glucose activity to that with native Con A is obtained (relGA=2.2 vs. 2.6) (see table 3).

TABLE 3 Influence of the degree of labeling (DOL) DOL Con A 1 1 Typenative pegylated Solution, rel GA 1 0.43 c = 0.5 mg Con A/g Matrix, relGA 2.55 2.22 c = 10 mg Con A/g

Through the pegylation, it first becomes possible to equip Con A with ahigh degree of labeling with a long wavelength fluorescent dye which isin the long-term stable in solution and does not precipitate.Surprisingly, no worsening of the glucose activity due to the pegylationis observed in enriching matrix compared to in solution. Hence, onlythrough the positive effect of the matrix is it possible to use PEG-ConA with a high degree of labeling at high concentrations in an assay.

Astonishingly, the glucose activity can be further increased byincreasing the degree of labeling on the Con A. In the enrichinghydrogel matrix, with Con A with DOL=2.5, almost a doubling of theglucose activity is achieved (rel GA=4.3 vs. 2.6). In direct comparisonto the measurement in solution, the glucose activity in matrix is thuseven increased 4.3-fold through the combined effect of concentration andDOL (see table 4).

TABLE 4 Influence of the degree of labeling (DOL) DOL Con A 1 1 2.5* 2.5Type native pegylated native* pegylated Solution, rel GA 1 0.43 1.03 c =0.5 mg Con A/g Matrix, rel GA 2.55 2.22 4.28 c = 10 mg Con A/g *nativeATTO680-Con A with a DOL of >1.5 is not stable in solution andprecipitates.

Example 6 Determination of the Influence of the Fluorescent Dyes

The structure of the fluorescent dyes which are bound to glucose-bindingprotein and ligand respectively also has an influence on the glucoseactivity. A combination of a rhodamine and an oxazine dye has provedparticularly suitable. For the Con A-dextran system, a rhodamine donor(e.g. ATTO590- or ATTO610-dextran or ROX-dextran) and an oxazineacceptor (e.g. ATTO655- or ATTO680-Con A, or Evoblue30-Con A) isparticularly preferable. Thereby, in contrast to the conventionally usedTMR-Con A/FITC-dextran system (xanthene-rhodamine combination), a2.1-fold glucose activity in solution and even 2.6-fold in enrichingmatrix is obtained. With a rhodamine-oxazine dye pair, a heterodimerinteraction can arise between the dyes which intensifies the glucoseactivity (see tables 5 and 6).

TABLE 5 Influence of the dyes Con A-dye ATTO680 TMR Dextran-dye ATTO590FITC DOL Con A 2.5* 2.3 Type native* native Solution, rel GA 2.4 1.17 c= 0.5 mg Con A/g Relative activity compared to the 2.05 1 conventionalsystem FITC/TMR Matrix, rel GA 4.92 1.9 c = 10 mg Con A/g Relativeactivity compared to the 2.59 1 conventional system FITC/TMRCorresponding values extrapolated on the basis of the experimentalvalues with PEG-Con A: FITC-dextran with 0.01 mol D/mol SU, mass ratiodex:Con A = 1:10

TABLE 6 Influence of the dyes ATTO590/ATTO680 ROX/Evoblue30 Matrix, relGA 2.55 2.1 c = 10 mg Con A/g Native Con A with DOL = 1, dextran with0.001 mol dye (D) per mol dex subunit (SU), mass ratio dex:Con A = 1:10

Example 7 Determination of the Influence of the Nature of the HydrogelMatrix

The glucose activity is also dependent on the nature of the hydrogelmatrix. The hydrogel matrix can consist of one polymer or of a mixtureof several polymers. A hydrogel matrix made of alginate or a mixture ofan alginate and a polyvinyl alcohol hydrogel has proved advantageous.Through its interaction with the Con A and the dextran, the alginatehydrogel enables the enrichment of the sensor components in highconcentrations.

The prepolymer of the 2^(nd) hydrogel (e.g. PVA) can penetrate into thealginate beads and after its crosslinking form an interpenetratingnetwork with the alginate. The mesh width of the interpenetratingnetwork influences the mobility of the molecules of the glucose-bindingprotein and of the ligand and thus also the glucose activity. The meshwidth can be influenced through the nature, the molecular weight and thesolids content of the polymer and the content of crosslinker groups,which determines the number of junctions.

A lower content of crosslinker groups leads to a higher glucoseactivity. Through a halving of the crosslinker groups, the activity caneven be increased 1.5-fold. Hence, compared to the system in solution, a4.2-fold improvement in the glucose activity can also be achievedwithout an increase in the degree of labeling on the Con A (see table7).

TABLE 7 Influence of the crosslinker content Crosslinker content(mmol/g) 0.486 0.33 0.26 Rel GA 2.76 4.04 4.24 Native Con A (13 mg/g)with DOL = 1, dextran with 0.001 mol dye (D) per mol dex subunit (SU),mass ratio dex:Con A = 1:10

The solids content of the network also has an influence on the glucoseactivity. The higher the solids content, the lower is the glucoseactivity (see table 8).

TABLE 8 Influence of the solids content SC Prepolymer 25% 30% 35% 40%Rel. GA 2.87 2.76 2.48 1.96 Native Con A (13 mg/g) with DOL = 1, dextranwith 0.001 mol dye (D) per mol dex subunit (SU), mass ratio dex:Con A =1:10

The invention claimed is:
 1. A method for enriching a dissolvedglucose-binding protein in a hydrogel, comprising: (a) providing apolymer-based hydrogel comprising at least one water-soluble polymer,wherein the polymer-based hydrogel comprises a first hydrogel matrix anda second hydrogel matrix which forms an interpenetrating network withinthe first hydrogel matrix; and (b) incorporating a glucose-bindingprotein and a ligand binding to the glucose-binding protein into thepolymer-based hydrogel of step (a), wherein (i) the at least onewater-soluble polymer is capable of interacting with the glucose-bindingprotein, and (ii) the glucose-binding protein is present in a complexwith the ligand, thereby enriching the dissolved glucose-binding proteinin the hydrogel.
 2. The method of claim 1, wherein the ligand of theglucose-binding protein is an oligosaccharide, a glycosylatedmacromolecule, or a glycosylated nanoparticle.
 3. The method of claim 1,wherein the dissolved glucose-binding protein is present at an at least5-fold, at least 10-fold, at least 15-fold, or at least 20-fold elevatedconcentration as compared to an aqueous solution.
 4. The method of claim1, wherein the at least one polymer is selected from the groupconsisting of alginates, sepharose, hyaluronic acid, chitosan, andcaragenans.
 5. The method of claim 1, wherein the polymer-based hydrogelcomprises a first hydrogel matrix comprising alginate and a secondhydrogel matrix which forms an interpenetrating network within the firsthydrogel matrix.
 6. The method of claim 1, wherein the glucose-bindingprotein is selected from the group consisting of lectins, enzymes whichbind glucose as substrate, antibodies which specifically recognizeglucose, and aptamers which specifically recognize glucose.
 7. Themethod of claim 1, wherein the glucose-binding protein is concanavalinA.
 8. The method of claim 7, wherein the concanavalin A concentration isgreater than about 0.5 mg/(g polymer).
 9. The method of claim 8, whereinthe concanavalin A concentration lies between about 2 and about 60 mg/(gpolymer).
 10. The method of claim 7, wherein the concanavalin A exhibitsincreased water solubility as compared to unmodified concanavalin Aowing to chemical modification.
 11. The method of claim 10, wherein themodification is selected from the group consisting of: pegylation,acetylation, succinylation, and polyoxazolinylation.
 12. The method ofclaim 2, wherein the glucose-binding protein is linked to an oxazine dyeand the ligand of the glucose-binding protein is linked to a rhodaminedye.